A comparative study of real-time RT-PCR based SARS-CoV-2 detection methods and its application to human derived and surface swabbed material

Real-time reverse transcription polymerase chain reaction (RT-PCR) remains a gold standard in detection of various viral diseases. In the COVID-19 pandemic, multiple RT-PCR based tests were developed to screen for viral infection. As an emergency response to growing testing demand, we established a SARS-CoV-2 PCR diagnostics platform for which we compared different commercial and in-house RT-PCR protocols. We evaluated four commercial (CDC 2019-nCoV, Applied Biosystems 2019-nCoV Assay Kit v1 TF-SinglePlex, 2019-nCoV Assay Kit v2 TF-MultiPlex, and EURORealTime SARS-CoV-2), one customized (Institute Pasteur), and one in-house RT-PCR protocols with 92 SARS-CoV-2 positive and 92 SARS-CoV-2 negative samples. Furthermore, we compared economical and practical characteristics of these protocols. We also developed a highly sensitive digital droplet PCR (ddPCR) method. Finally, we conducted a local environmental study for the presence and infectivity of SARS-CoV-2 on different surfaces in a quarantined household using RT- and ddPCR methods. We found very low limits of detection (1 or 2 viral copies/L), high sensitivities (93.6-97.8%) and specificities (98.7-100%) for the tested RT-PCR protocols. We further demonstrated the feasibility of downscaling two of the commercial protocols, which could optimize testing capacity. In the local environmental study, only one surface sample tested positive for viral RNA, but without detectable infectivity in vitro. Tested commercial and customized RT-PCR detection kits show very good and comparable sensitivity, and specificity, and the kits could be further optimized for use on SARS-CoV-2 viral samples derived from human and surface swabbed samples.

Comparison of RNA extraction methods for the detection of SARS-CoV-2 by RT-PCR

ObjectivesThe SARS-CoV-2 pandemic outbreak has stressed health care systems as well as medical supply chains, but diagnostic testing is an essential public health measure to control viral spread. Here we test the suitability of different RNA extraction methods for integration into a diagnostic workflow for coronavirus testing. MethodsWe applied six RNA extraction methods on the same 24 SARS-CoV-2 positive patient samples and quantified their results by subsequent reverse-transcriptase PCR (RT-PCR) of three viral genes. These methods included a) column-based extraction, b) phenol-chloroform extraction, as well as c) extraction using magnetic beads (i.e., one commercial kit as well as three different magnetic beads in combination with home-brewed buffers and solutions). ResultsWe achieved diagnostic-quality RT-PCR results with all methods, and there was no significant difference between the tested methods, except for one magnetic bead protocol with home-brewed buffers, in which the number of positive tested genes was significantly lower. ConclusionsFive of the six RNA extraction methods are interchangeable in a diagnostic workflow. Since some methods are more scalable than others, and have comparable results on RT-PCR quantitation, they may be more amenable to high-throughput sample processing pipelines. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/20172494v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@f9ff33org.highwire.dtl.DTLVardef@e17c21org.highwire.dtl.DTLVardef@19c8c37org.highwire.dtl.DTLVardef@b9aa2d_HPS_FORMAT_FIGEXP M_FIG C_FIG